Mycobacterium tuberculosis (M. tuberculosis) is a major pathogen of global importance. Despite the availability of chemotherapy and the Bacille Calmette-Guerin vaccine, M. tuberculosis continues to claim more lives than any other single infectious agent. Recent years have seen increased incidence of tuberculosis in both developing and industrialized countries, the widespread emergence of drug-resistant strains and a deadly synergy with the human immunodeficiency virus. The limitation of current therapy originates from the widespread occurrence of antibiotic resistant strains and also the requirement for prolonged and uninterrupted administration of antibiotics. Thus, new classes of antibiotics are desperately needed. Sulfur-containing metabolites, including the essential amino acids cysteine and methionine, and the virulence factors mycothiol and sulfolipid-1, are crucial for the infectivity of M. tuberculosis. Their biosynthesis involves the sulfate assimilation pathway, which has no counterpart in humans. Since the enzymes in this pathway comprise a central hub of metabolism and are unique to bacteria, we propose that these enzymes are prime targets for anti-mycobacterial therapy. This proposal focuses on 5'-adenosinephosphosulfate (APS) reductase (CysH) as a target for small molecule drug development. In preliminary work, we have 1) confirmed the APS reductase activity of M. tuberculosis CysH using genetic complementation in E. coli, and 2) demonstrated that genetic deletion of CysH from a pathogenic strain of M. tuberculosis renders the mutant unable to establish infection in a mouse model of tuberculosis. In specific aim 1 of this proposal we shall screen a compound library for inhibitors of M. tuberculosis sulfate assimilation using an E. coli complementation assay. In specific aim 2 we shall set up cell-free homogenous assays for recombinant M. tuberculosis CysH, examine our leads from the primary screen for their ability to directly inhibit this enzyme, and optimize these leads through medicinal chemistry. In specific aim 3 the best compounds will be examined using in vitro susceptibility studies of clinical isolates of M. tuberculosis. In phase II of this study, the inhibitor leads from our feasibility study will be examined for bacteriostatic and bacteriocidic efficacy in mouse models of tuberculosis. The long-term objective of this study is to develop novel antibiotics for more effective treatment of human tuberculosis and other mycobacterial diseases.